Kan Shida

Lactobacillus casei Inhibits Antigen-Induced IgE Secretion through Regulation of Cytokine Production in Murine Splenocyte Cultures

Background:Lactobacillus casei is a nonpathogenic gram-positive bacterium widely used in dairy products and has been shown to enhance the cellular immunity of the host. Methods: To examine the inhibitory effect of L. casei on IgE production, splenocytes obtained from ovalbumin (OVA)-primed BALB/c mice were restimulated in vitro with the same antigen in the presence of heat-killed L. casei. The effect of this bacterium on T helper (Th) phenotype development was also examined with naive T cells from OVA-specific T cell receptor-transgenic mice. Results:L. casei induced IFN-γ, but inhibited IL-4 and IL-5 secretion, and markedly suppressed total and antigen-specific IgE secretion by OVA-stimulated splenocytes. The inhibitory effect of L. casei on IgE, IL-4, and IL-5 production was partially abrogated by addition of neutralizing antibody to IFN-γ. Augmented IL-12 production was also observed in the cell cultures containing L. casei, and anti-IL-12 monoclonal antibody completely restored the IgE, IL-4, and IL-5 production to the control levels. The IL-12 augmentation by L. casei was macrophage-dependent. The Th cell development assay showed the ability of L. casei to induce Th1 development preferentially. This effect was also completely blocked by anti-IL-12 antibody. Conclusions: This is the first demonstration that a nonpathogenic microorganism, L. casei, can inhibit antigen-induced IgE production through induction of IL-12 secretion by macrophages. The findings suggest a potential use of this organism in preventing IgE-mediated allergy.

Effects of Probiotics on Allergic Rhinitis Induced by Japanese Cedar Pollen: Randomized Double-Blind, Placebo-Controlled Clinical Trial

Background:Lactobacillus casei strain Shirota (LcS) has been found to exert antiallergic effects in animal experiments, but there is little information about its clinical effects in human patients with allergy. Methods: We performed a randomized double-blind, placebo-controlled study to investigate the effects of LcS in patients with allergic rhinitis triggered by Japanese cedar pollen (JCP). Participants were asked to drink fermented milk containing LcS (LcS group) or placebo (control group) for 8 weeks. Clinical symptoms and immunological parameters were compared between the two groups. Results: Symptom-medication scores (SMS) worsened in accordance with the increase in the amount of scattered JCP. In terms of the nasal and ocular SMS, there was no significant difference between the LcS group and the placebo group during the ingestion period. In the subgroup of patients with moderate-to-severe nasal symptom scores before starting the ingestion of test samples, supplementation with LcS tended to reduce nasal SMS. Conclusion: These results indicate that fermented milk containing LcS does not prevent allergic symptoms in patients sensitive to JCP, but may delay the occurrence of allergic symptoms in patients with moderate-to-severe nasal symptom scores.

Probiotics and immunology: separating the wheat from the chaff

Probiotics are live bacteria exhibiting health-promoting activities. Recent research has demonstrated that probiotics can prevent pathogen colonization of the gut and reduce the incidence or relieve the symptoms of various diseases caused by dysregulated immune responses. Probiotics seem to function by influencing both intestinal epithelial cells and immune cells of the gut, but the details of these effects are still being unraveled. Therefore, probiotics, through their effects on the host immune system, might ameliorate diseases triggered by disordered immune responses. Caveats remain and, because the beneficial effects of probiotics can vary between strains, the selection of the most suitable ones will be crucial for their use in the prevention or treatment of specific diseases.

Peptidoglycan from lactobacilli inhibits interleukin‐12 production by macrophages induced by Lactobacillus casei through Toll‐like receptor 2‐dependent and independent mechanisms

We previously showed that Lactobacillus strains having a rigid cell wall resistant to intracellular digestion can stimulate macrophages to induce large a quantity of interleukin‐12 (IL‐12). In this study, we examined the influence of lactobacilli and bacterial cell wall components on IL‐12 production by macrophages that was induced by Lactobacillus casei, which has a rigid cell wall. Easily digestible lactobacilli such as Lactobacillus johnsonii and Lactobacillus plantarum or their intact cell walls (ICWs) weakly or very weakly induced IL‐12 production by macrophages, and inhibitedL. casei‐induced IL‐12 production. While the ICW of L. casei was resistant to intracellular digestion and did not inhibit L. casei‐induced IL‐12 production, its polysaccharide‐depleted ICW, i.e. intact peptidoglycan, was sensitive to intracellular digestion and inhibited L. casei‐induced IL‐12 production. Furthermore, the peptidoglycans of L. johnsonii, L. plantarum and Staphylococcus aureus also inhibited L. casei‐induced IL‐12 production. Peptidoglycans from lactobacilli suppressed L. casei‐induced expression of IL‐12p40 but not IL‐12p35 mRNA. Inhibition of IL‐12 production by peptidoglycan was mitigated in Toll‐like receptor 2 (TLR2)‐deficient macrophages compared with the inhibition in wild‐type macrophages. A derivative of the minimal structural unit of peptidoglycan (6‐O‐stearoyl‐muramyl dipeptide) recognized by nucleotide‐binding oligomerization domain 2 (NOD2) could also suppress L. casei‐induced IL‐12 production. These findings demonstrate that easily digestible bacteria and peptidoglycan suppress IL‐12 production through pattern recognition receptors such as TLR2 and NOD2. IL‐12 production in the gut may be negatively regulated by the simultaneous inhibitory actions of various resident bacteria that are susceptible to intracellular digestion.

Oral administration of probiotic bacteria, Lactobacillus casei and Bifidobacterium breve, does not exacerbate neurological symptoms in experimental autoimmune encephalomyelitis

To evaluate the safety of two probiotic bacterial strains, Lactobacillus casei strain Shirota (LcS) and Bifidobacterium breve strain Yakult (BbY), these probiotics were orally administered to Lewis rats with experimental autoimmune encephalomyelitis (EAE), the experimental model of human multiple sclerosis. We examined three experimental designs by combining different antigen types and probiotic administration periods: (1) EAE was induced with a homogenate of guinea pig spinal cord as the sensitizing antigen, and LcS was orally administered from one week before this sensitization until the end of the experiment; (2) EAE was induced using guinea pig originated myelin basic protein (MBP) as the sensitizing antigen, and LcS was orally administered from one week before this sensitization to the end of the experiment; (3) EAE was induced using guinea pig MBP as the sensitizing antigen, and the probiotic strains (LcS and BbY) were administered starting in infancy (two weeks old) and continued until the end of the experiment. In experiment 1, oral administration of LcS tended to suppress the development of neurological symptoms. Differences in neurological symptoms between the control group and the administration groups did not reach statistical significance in experiments 2 and 3. These results support the notion that neither LcS nor BbY exacerbates autoimmune disease.

Probiotic upregulation of peripheral IL-17 responses does not exacerbate neurological symptoms in experimental autoimmune encephalomyelitis mouse models

Context: It is of great importance to evaluate the safety of probiotics in dysregulated immune conditions, as probiotics can possibly modulate immune functions in the host. Objective: We tried to confirm the safety of using Lactobacillus casei strain Shirota (LcS) to help prevent autoimmunity in the central nervous system. Methods: We used two chronic experimental autoimmune encephalomyelitis (EAE) models, a relapse and remission type EAE model in SJL/J mice and a durable type model in C57BL/6 mice. LcS was administered from 1 week before antigen sensitization until the end of the experiments, and neurological symptoms and histopathological changes of the spinal cord were observed. Immunological parameters were also examined in the SJL/J mouse model. Results: LcS administration did not exacerbate neurological symptoms or histopathological changes of the spinal cord in either model but instead tended to improve neurological symptoms in the SJL/J mouse EAE model. LcS administration transiently upregulated IL-17 production by antigen-stimulated lymphocytes of draining lymph nodes 7 days after sensitization. Enhanced production of IL-10 and an increase in the percentage of CD4+CD25+ T regulatory cells were also observed at the same sites. Strong expression of IL-17 mRNA was detected in the spinal cord of mice that displayed severe neurological symptoms on day 12, but this expression was not enhanced by LcS administration. Conclusion: These results demonstrate that LcS does not exacerbate, but instead may improve EAE depending on the immunization conditions, and that IL-17 responses at peripheral sites may not always result in a worsening of autoimmune diseases.

Microbiota of the Small Intestine Is Selectively Engulfed by Phagocytes of the Lamina Propria and Peyer’s Patches

Phagocytes such as dendritic cells and macrophages, which are distributed in the small intestinal mucosa, play a crucial role in maintaining mucosal homeostasis by sampling the luminal gut microbiota. However, there is limited information regarding microbial uptake in a steady state. We investigated the composition of murine gut microbiota that is engulfed by phagocytes of specific subsets in the small intestinal lamina propria (SILP) and Peyer’s patches (PP). Analysis of bacterial 16S rRNA gene amplicon sequences revealed that: 1) all the phagocyte subsets in the SILP primarily engulfed Lactobacillus (the most abundant microbe in the small intestine), whereas CD11bhi and CD11bhiCD11chi cell subsets in PP mostly engulfed segmented filamentous bacteria (indigenous bacteria in rodents that are reported to adhere to intestinal epithelial cells); and 2) among the Lactobacillus species engulfed by the SILP cell subsets, L. murinus was engulfed more frequently than L. taiwanensis, although both these Lactobacillus species were abundant in the small intestine under physiological conditions. These results suggest that small intestinal microbiota is selectively engulfed by phagocytes that localize in the adjacent intestinal mucosa in a steady state. These observations may provide insight into the crucial role of phagocytes in immune surveillance of the small intestinal mucosa.

Beneficial Effects of Citrus Juice Fermented with Lactobacillus plantarum YIT 0132 on Japanese Cedar Pollinosis

Recently, the prevalence of allergies in Japan has been increasing. Certain types of fruit juice and lactic acid bacteria are known to alleviate allergic symptoms. Therefore, we examined whether citrus juice fermented by a specific lactic acid bacteria can improve the symptoms of Japanese cedar pollinosis (JCPsis). Lactobacillus plantarum YIT 0132 (LP0132) was selected based on its high proliferative activity in citrus juice and anti-inflammatory interleukin-10-inducing activity. Dietary administration of heat-killed LP0132 cells or citrus juice fermented with LP0132 was found to significantly suppress nasal rubbing in a JCPsis mouse model, indicating relief of allergy symptoms. To evaluate the effects of LP0132-fermented citrus juice on pollinosis symptoms and quality of life (QOL) in humans with JCPsis, a single-blind, placebo-controlled, parallel-group clinical trial was conducted. The participants were 42 adults with JCPsis. They ingested 100 mL of sterilized LP0132-fermented citrus juice (active group) or unfermented citrus juice (placebo group) once daily for 8 weeks. Immediately after the pollen peak when allergy symptoms and QOL loss were most severe, itchy eyes, itchy skin, and QOL loss by JCPsis were alleviated in the active group compared with the placebo group. At 10 weeks after starting the intervention, increased the levels of blood eosinophils were significantly suppressed in the active group compared with the placebo group. We conclude that continuous ingestion of citrus juice fermented with LP0132 may help alleviate the allergy symptoms and impaired QOL caused by JCPsis.

Escherichia coli Heat-Labile Enterotoxin Binds to Glycosylated Proteins with Lactose by Amino Carbonyl Reaction

The binding of Escherichia coli heat‐labile enterotoxin (LT) type I to glycosylated proteins with lactose (Galβ1‐4Glc) by amino carbonyl reaction was studied by the Western blot assay and by the microtiter well binding assay. LT bound to a lactose‐α‐lactalbumin amino carbonyl product (Lac‐LA), whereas cholera toxin did not. The binding ability of Lac‐LA was abolished by β‐galactosidase treatment, indicating that the terminal galactose is essential for the binding of LT. The binding of LT to Lac‐LA was inhibited by galactose and lactose, and most effectively inhibited by lactulose (Galβ1‐4Fru), which is a structural analog of the Amadori rearrangement product of the amino carbonyl reaction between lactose and an ε‐amino group of a lysine residue (lactuloselysine). The results suggest that LT recognizes the portion of lactuloselysine in Lac‐LA. LT also bound to a melibiose (Galα1‐6Glc)‐α‐lactalbumin amino carbonyl product (Mel‐LA), but the binding ability of Mel‐LA was weaker than that of Lac‐LA, suggesting that the β1‐4 linked terminal galactose is dispensable but preferable for the binding. Furthermore, LT bound to the amino carbonyl products of lactose with β‐lactoglobulin, caseins, bovine serum albumin, and ovalbumin. These results indicate that LT binds to the amino carbonyl products between proteins and sugars containing the terminal galactose, such as lactose.

Enhanced differentiation of intraepithelial lymphocytes in the intestine of polymeric immunoglobulin receptor-deficient mice

To clarify the effect of secretory IgA (sIgA) deficiency on gut homeostasis, we examined intraepithelial lymphocytes (IELs) in the small intestine (SI) of polymeric immunoglobulin receptor‐deficient (pIgR−/−) mice. The pIgR−/− mice exhibited the accumulation of CD8αβ+ T‐cell receptor (TCR)‐αβ+ IELs (CD8αβ+αβ‐IELs) after weaning, but no increase of CD8αβ+γδ‐IELs was detected in pIgR−/− TCR‐β−/− mice compared with pIgR+/+ TCR‐β−/− mice. When 5‐bromo‐2′‐deoxyuridine (BrdU) was given for 14 days, the proportion of BrdU‐labelled cells in SI‐IELs was not different between pIgR+/+ mice and pIgR−/− mice. However, the proportion of BrdU‐labelled CD8αβ+‐IELs became higher in pIgR−/− mice than pIgR+/+ mice 10 days after discontinuing BrdU‐labelling. Intravenously transferred splenic T cells migrated into the intraepithelial compartments of pIgR+/+ TCR‐β−/− mice and pIgR−/− TCR‐β−/− mice to a similar extent. In contrast, in the case of injection of immature bone marrow cells, CD8αβ+αβ‐IELs increased much more in the SI of pIgR−/− TCR‐β−/− mice than pIgR+/+ TCR‐β−/− mice 8 weeks after the transfer. αβ‐IELs from pIgR−/− mice could produce more interferon‐γ and interleukin‐17 than those of pIgR+/+ mice, and intestinal permeability tended to increase in the SI of pIgR−/− mice with aging. Taken together, these results indicate that activated CD8αβ+αβ‐IELs preferentially accumulate in pIgR−/− mice through the enhanced differentiation of immature haematopoietic precursor cells, which may subsequently result in the disruption of epithelial integrity.